Project Summary/Abstract Neuroinflammation is increasingly thought of as playing a causative role in neurodegenerative disease, with evidence of inflammatory processes preceding neuronal death. For instance, microglia are now thought of as crucial participants in the onset of diseases such as Alzheimer?s disease (AD) and Parkinson?s disease (PD). This shift in focus from neurons to microglia has made evident a glaring need for tools which allows for precise manipulation of microglia. In the last decades there has been great progress in the generation of tools aimed at studying brain form and function. However, despite these advances, barriers remain. Despite the crucial role of microglia in disease, tools to study their function and to manipulate their expression (in disease) are lacking. Certainly the use of inducible transgenic animals has represented a significant step forward. Still, such approaches have limits. For instance, CRE-based models do not facilitate the precise modulation of multiple cellular populations at the same time. To that end, we contend that viral vectors will represent an integral part in studying the role of microglia in disease, and that the use of vectors that can target non-neuronal cells with efficacy and fidelity will be a requisite and a significant advance in translational neurodegenerative research. Thus far, viral vectors have proven to be remarkably refractory to microglial transduction. Thus, herein we propose a novel approach whereby we will engineer novel vectors directed towards microglia with no off-target activity. Our preliminary data using AAV show that there is no biological reason for this apparent impediment in microglial infection. In a single Aim we will utilize an innovative workflow where we will insert a library of known microglial ligands into the capsid of AAV, and where each variant will be unequivocally linked to a unique genetic barcode. This library will be injected in to the brain of mice, and single cell RNAseq (scRNAseq; also identifying the barcode) will be performed. Using genomics we will thus be able to associate each unique barcode/capsid variant with one, or multiple, cellular transcriptional profiles. Importantly, this will allow us to perform in silico negative selection to screen for capsids that only transduce microglia. We will validate the novel viral vectors in aged and inflamed animals, and also demonstrate the utility of this microglial-specific vector in a the 5XFAD mouse model of AD, where we will test the hypothesis that inhibiting Toll-like receptor 4 in microglia per se is sufficient to prevent neuroinflammation and neurodegeneration. At the completion of this proposal we will have generated a novel tool to help us manipulate microglial function with precision, and under conditions that is reflective of the degenerating brain. Thus providing for a new and crucial foundation to move translational efforts within neurodegeneration forward. Moreover, we will also have generated a unique database of capsid variants and their associated transduction transcriptome pattern. This database can be queried by any researcher in order to identify vectors that transduce other cells, perhaps even subsets of neuronal cells. These novel tools will have far-reaching impact on the field of neurodegeneration.